Metagenomic sequencing for identifying pathogen-specific circulating DNAs and development of diagnostic methods for schistosomiasis

Summary Timely diagnosis of Schistosoma infection, particularly in the early stage is crucial for identifying infected hosts and then taking effective control strategies. Here, metagenomic next-generation sequencing was used to identify pathogen-specific circulating DNAs (cDNAs) in the sera/plasma of New Zealand rabbits infected with S. japonicum, and the identified cDNAs were validated by PCR and qPCR. Loop-mediated isothermal amplification (LAMP)-based CRISPR-Cas12a and recombinase polymerase amplification-based lateral flow strip (RPA-LF) methods combined with the newly identified cDNA were developed to evaluate the potentials for diagnosing murine and human schistosomiasis. The results indicated that twenty-two cDNAs were identified. The developed LAMP-based CRISPR/Cas12a and RPA-LF methods showed a good potential for diagnosing murine or human schistosomiasis as early as 5 days of post-infection with 5 cercariae infection. In a word, S. japonicum specific cDNAs in circulation of infected hosts could be effective biomarkers for detecting Schistosoma infection particularly for early stages.


INTRODUCTION
Schistosomiasis is a devastating parasitic disease that affects more than 200 million people worldwide. 1 Despite remarkable progress in controlling the disease through snails elimination, mass drug administration, and generally combined strategies in China over the past 70 years, 2 the prevalence of schistosomiasis and the risk of infection still remain an important health problem in some areas. 3,4 The widely used method for diagnosing involves microscopy-based detection of parasite eggs in urine (S. haematobium) or feces (S. mansoni and S. japonicum), 5 which have relatively low-throughput, time-consumption, skilled personnel is required for operation, limiting its diagnostic value in endemic regions. 6 Additionally, it usually causes an underestimation of the prevalence and infection of this disease due to the results depending on the efficiency of egg-shedding that varies from day to day. 7 Consequently, sensitive and effective monitoring and/or diagnostic strategies are greatly needed for schistosomiasis control and elimination. 8 Serological methods for detecting Schistosoma infection offers relatively more sensitive and less timeconsuming than the microscopy method. However, current serological tests using antigens isolated from eggs or adult worms suffer from lowering specificity and increasing false-positive results. 9 Alternatively, purified recombinant proteins were suggested to be better antigens for schistosomiasis diagnosis such as saposin-like proteins (SjSAPLP4, SjSAPLP5, and SjSP13), 10,11 multidrug-resistant protein 1-encoding gene (SjMRP1), 12 and Sm-SLP-1 13 that showed improved specificity. However, the antibodies recognized by these antigens can only be detected in the late stage of Schistosoma infection (3-4 weeks after infection), 10 lack of the capability of detection for early stages. In addition, serological methods are unable to distinguish between ongoing and previous infections. 14 Recently, nucleic acid-based molecular diagnosis for schistosomiasis was developed and showed high sensitivity and specificity, which are considered to be an attractive diagnostic approach. 15 Although these methods can detect schistosomiasis in snails, contaminated waters, fecal samples of animals or patients, [16][17][18][19][20][21] the results are largely dependent on the number of eggs or parasites presented in samples used for DNA extraction. Circulating free-cell DNAs (cDNAs), found in blood and other body fluids of mammals have been identified as novel biomarkers for diagnosing different clinical conditions. 22 Recently, cDNAs derived from some tumors can be used for cancer diagnosis 23,24 and cDNAs in maternal plasma can be used for diagnosis of pregnancy and pregnancy-associated complications. 25 More recently, the usage of parasitic DNA has been successfully validated in the diagnosis of parasites, such as Plasmodium, 26 Leishmania, 27 and S. mansoni. 28 In S. japonicum, polymerase chain reaction (PCR), real-time PCR, and loopmediated isothermal amplification (LAMP) based on molecular detection were also shown to perform relatively high sensitivity in both animal and human schistosomiasis. [29][30][31][32] Adult Schistosoma lives in the blood vessels of final hosts. During the parasitic migration, worm development, and interaction with host immune response, parasitic cells and/or tissues may be turnover and release cDNAs. The cDNA could be distributed throughout the circulation of hosts, which provides an ideal live biomarker for detecting Schistosoma infection. Therefore, we performed metagenomic highthroughput sequencing to systematically identify Schistosoma-specific cDNAs from the circulation of infected hosts and then developed LAMP-CRISPR/Cas12a and RPA-LF methods for evaluating the potential of identified cDNA for detecting S. japonicum infection.

RESULTS
Metagenomic next-generation sequencing for identification of pathogen-specific cDNAs in the circulation of rabbits infected with S. japonicum In total, twenty-two S. japonicum-specific cDNAs were identified (Figures 1, S2, and S3, and Table S1). Blast analysis indicated that three cDNAs were matched to SJCHGC08404 protein mRNA (AY812939.1: k141_50, k141_54, and k141_59); four cDNAs were related to non-LTR retrotransposon SjCHGCS19 (FN356221.1: k141_3, 17, 49, and 63); six cDNAs were related to non-LTR retrotransposon SjR2-like sequence (AF412215.1: k141_15, 25, 29, 134; AF412216.1: k141_77 and AF412220.1: k141_143); two cDNAs were (B) Binformatic workflow and results for metagenomic sequencing data. cDNA was isolated from serum samples of rabbits infected with approximately 200-500 cercariae. Following the quality control, the cDNA was fragmented to construct a DNA library that was subsequently sequenced using the Illumina HiSeq platform. The raw data were cleaned and mapped to rabbit genome. After removing reads mapped to the rabbit genome, the other reads were mapped to S. japoniucm genome. S. japonicum cDNA sequences were obtained by filtering the reads were smaller than 140 bp. Further de novo assembly and blast analysis identified twenty-two cDNA sequences.

Verification of S. japonicum cDNAs in sera of infected mice
To verify the identified cDNAs consistently presented in the circulation of hosts regardless of different species, we designed the PCR primers for 22 identified S. japonicum cDNAs and used mice as an animal model to verify the metagenomic next-generation sequencing results. Upon PCR amplification and agarose gel analysis, we found that 12 cDNAs, including K141_1, K141_3, K141_39, K141_49, K141_15, K141_25, K141_29, K141_63, K141_74, K141_77, K141_134, and K141_90 were detected in sera of S. japonicum-infected mice while there was no detection in the sera of uninfected animals and blank controls (Figures 2A and S1). To further validate the results, we used qPCR to determine the amount of cDNAs in the circulation of hosts, the results indicated that the cDNAs can be consistently presented and SjR2LS showed the highest amount ( Figure 2B). Similar results were also to some extent observed in the experiments with spiked-in control ( Figure S4).
Evaluation of the diagnostic potential of SjR2LS and S20 for S. japonicum infection SjR2-like sequence (SjR2LS, k141_29) and S20 (K141_74) were selected to further investigate the potential of SjR2LS for diagnosing schistosomiasis. Sera from infected mice (n = 20) were subjected to PCR analysis and ELISA analysis. SjR2LS was detected in all of sera samples collected from mice infected with S. japonicum while relatively less number of infected mice was observed when applying S20 ( Figures 3A and S1). ImageJ analysis of Figure 3A for performing ROC analysis indicated the diagnostic value of SjR2LS (AUC = 1 for SjR2LS) is better than that of S20 (AUC = 0.9025 for S20) ( Figure 3B). Sera collected from mice at 30 days of post S. japonicum infection as well as uninfected mice were analyzed by using ELISA method. The results indicated the diagnostic accuracy of ELISA (AUC = 0.985) ( Figure 3C) is less sensitive than that of PCRbased SjR2LS ( Figure 3D).

PCR-based SjR2LS method is more sensitive than ELISA method
To further confirm the sensitivity of SjR2LS for detecting S. japonicum infection, twenty-four mice were randomly divided into four groups including one uninfected group and three infected groups with different 10, 20, 40 cercariae inoculations, respectively. Sera were collected from infected mice at 5, 10, 15, 20, 25, and 30 days of post-infection, respectively. Worm burden in each mouse was also observed at 30 days of post-infection. SjR2LS was determined by PCR from sera collected from mice at 30 days of post-infection. The results indicated that SjR2LS was able to be detected in the cDNAs isolated from sera samples of mice that perfused to collect at least 5-7 worms (Figures 4A and S1) while similar results were also observed using ELISA method ( Figure 4B). Importantly, we observed that the detectable capacity using SjR2LS in infected mice with 5 worms can be observed as early as 5 days of post-infection ( Figure 4C) while ELISA method was showed the credible detection at least at 25 days of post-infection ( Figure 4D). These results indicated that detection of S. japonicum infection using SjR2LS-based PCR is more sensitive than that of ELISA method for early stage.

LAMP-based CRISPR/Cas12a method for detecting S. Japonicum infection
Upon the optimized conditions for LAMP assay as well as the identification of digest activity for CRISPR/ Cas12a ( Figures S1 and S5), we developed a LAMP-based CRISPR/Cas12a combined with the fluorescent reporter to increase the sensitivity for detection. Upon analyses of plasmas collected from the uninfected control and infected mice at different days of post-infection (5 days, 7 days, and 9days), we found that the percentage of detection are 75%, 87.5%, and 100%, respectively ( Figures 5 and S1). The results indicated that the LAMP-based CRISPR/Cas12a method can to some extent detect S. japonicum infection as early as 5 days of post-infections.

RPA-LF method for detecting S. japonicum infection
Alternatively, we further developed a SjR2LS-based rapid polymerase amplification combined with a lateral flow strip (RPA-LF) assay. Firstly, positive serum samples isolated from mice infected with S. japonicum at 20 days of post-infection and uninfected serum samples were evaluated using the developed RPA-LF assay. As shown in Figure 6A, SjR2LS can be amplified by LF-RPA in the sera of infected mice, while no signal was observed in sera isolated from uninfected mice. As to the sensitivity of RPA-LF, we noted that SjR2LS was able to be detected in mice infected with 5 cercariae as early as 5 days of post-infection ( Figures 6B and S1). Furthermore, we evaluated the diagnostic performance of the SjR2LS-based RPA-LF for diagnosing schistosomiasis in human patients. As shown in Figure 6C, the positive signal for SjR2LS can be observed in 10 iScience Article out of 11 serum samples of human schistosomiasis, which is more sensitive than SjR2LS-based PCR method, that detect 9 out of 11.

DISCUSSION
Current methods for schistosomiasis diagnosis usually suffer from time-consuming, lack of sensitivity, or less capability of detection for early infection. Consequently, there is a great need to develop an effective method showing live parasites with high sensitivity for detecting Schistosoma infection, particularly for the early stage. 33 Since Schistosoma live in the blood vessels of the host, pathogen-specific cDNAs in the circulation of the infected hosts may provide a unique advantage for developing live-infection biomarkers for diagnosing schistosomiasis. Currently, nucleic acid-based methods such as PCR, real-time PCR, LAMP, and RPA have been successfully developed and implicated in diagnosing schistosomiasis. 28,32 The sensitivity and specificity of these methods are affected by various factors; one of the key factors is biomarkers. Several DNA biomarkers have been evaluated for schistosomiasis diagnosis including Dra1 sequence of S. haematobium (GenBank: DQ157698.1), 34 a 121 bp tandem repeated DNA sequence of S. mansoni, 28,35 and SjCHGCS19 and SjR2 of S. japonicum. 29,30,36 However, the effective biomarker for S. japonicum infection is still needed.
In the present study, we systematically identified cDNAs from plasma/sera of rabbits infected with S. japonicum, resulting in 22 cDNAs that matched to S. japonicum genome. The resulted cDNAs are not reported previously. Although a couple of cDNAs were shown to match some GenBank Gene IDs, such as SjCHGCS19 (GenBank: FN356221.1) that have been evaluated their diagnostic potential in a previous study. 30 Our findings indicated that different regions related to the genes are matched to the identified cDNA. Importantly, we identified six SjR2-like cDNAs, including AF412215.1 (k141_15, 25, 29, 134), AF412216.1 (k141_77), and AF412220.1 (k141_143), while SjR2 (GenBank: AF412221.1) detected in sera samples of animals infected with S. japonicum 29,30,36 was not identified in our present study. We noted there are different regions presented as cDNA for one gene, suggesting that cDNAs may be released in region-dependent manners.
Additionally, we further validate the identified cDNAs to be detectable in S. japonicum-infected mice sera, confirming that 12 out of 22 can serve as biomarkers for diagnosing murine schistosomiasis. Further iScience Article investigation of SjR2LS indicated that the detection rate of S. japonicum infection is 100% in mice infected with S. japonicum, which is more sensitive than the ELISA method. LAMP and RPA are more attractive for field application because the amplification can be done rapidly at a constant temperature and naked eyes can visualize the results. We combined LAMP with CRISPR/Cas12a to improve the sensitivity by using fluorescent labeled probes. The results indicated that LAMP-CRISPR/Cas12a-based method could detect S. japonicum infection as early as 5 days with 5 cercariae. Additionally, RPA has more advantages, such as being done at a significantly low temperature (37 C-42 C) and a rapid time (within 20 min). In previous studies, real-time RPA and LF-RPA assays targeting the SjR2 (AF412221.1) have been investigated to detect S. japonicum infection in the fecal or serum samples of patients. 37,38 Here, we documented an alternative SjR2LS and further developed two methods, including LAMP-CRISPR/Cas12a and LF-RPA. It was shown that SjR2LS can be amplified by LF-RPA assay in cDNAs isolated from serum samples of infected mice within 15 min. Furthermore, higher sensitivity of the established LF-RPA method was observed than the PCR method when implicated in diagnosing the serum samples from persons infected with S. japonicum.
In summary, metagenomic next-generation sequencing for identification of parasite-specific cDNAs associated with S. japonicum infection was carried out in the present study and twenty-two cDNAs were found and validated in different animal models. We also developed LAMP-CRISPR/Cas12a and LF-RPA methods combined with newly identified cDNA and applied to S. japonicum-infected animal and human samples successfully. These findings provided a resource for establishing sensitive molecular approaches for diagnosing schistosomiasis. The developed LAMP-CRISPR/Cas12a and LF-RPA assays may represent an alternative method for detecting Schistosoma infection, particularly for the early stage.

Limitations of the study
This study presents the identification of S. japonicum-specific cDNAs in plasma/sera rabbits and verification of some of these identified cDNAs by PCR, qPCR, and the developed LAMP-CRISPR/Cas12a and LF-RPA methods. However, some issues still require further investigation. Firstly, it is unclear about the relativity between the abundance of identified S. japonicum-specific cDNAs and different hosts. Secondly, due to the availability of human sera for schistosomiasis patients, we only evaluated 11 patients using the developed LF_RPA method in the present study. It is necessary to increase sample size to further validate the developed methods.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:   (B) Evaluation of detectable capacity of SjR2LS-based LF-RPA method. Twelve mice were randomly divided into two groups (infection and control) and each mouse in the infected group was infected with 5-10 cercariae. The sera from infected groups and controls were collected at indicated times as described in the method section and were further analyzed by LF-RPA. (C) Evaluation of diagnostic capacity of SjR2LS-based LF-RPA for human sera collected from human schistosomiasis. LF-RPA results showed that SjR2LS can be amplified in 10 out of 11 serum samples from human schistosomiasis japonica, which is more sensitive than the PCR method that detects 9 out of 11 samples. T indicated test samples; C indicated control for assay. iScience 26, 107495, September 15, 2023 iScience Article RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Guofeng Cheng (cheng_guofeng@foxmail.com or chengguofeng@tongji.edu.cn).

Materials availability
This study did not generate new unique reagents.
Data and code availability d The raw sequence data reported in this paper have been deposited in the NCBI SRA database: PRJNA982300.
d This paper does not report original code.
d The datasets are publicly available. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Animal experiments
The life cycle of S. japonicum (Anhui isolate) were maintained in New Zealand rabbits and Kunming mice and Oncomelania hupensis was obtained from the National Institute of Parasitic Disease, Chinese Center for Disease Control and Prevention (Shanghai, China). New Zealand rabbits with a weight of 2.5-3 Kg (male, for each group, n = 5) were infected with approximately 200-500 cercariae via abdominal skin penetration. For mice experiment I, two groups of eight-week-old male Kunming mice (for each group, n = 20) were used. One group of mice were challenged with approximately 40 cercariae while another group served as non-infected control. For mice experiment II, four groups of eight-week-old male mice (for each group, n = 6) were used. One group of non-infected mice were served as control, while the other three groups were challenged with 10, 20 and 40 cercariae, respectively. For mice experiment III, two groups of eight-weekold male Kunming mice (for each group, n = 8) were used and each mouse was challenged with 5 cercariae in infected group while another group served as non-infected control. For mice experiment IV, two groups of eight-week-old male Kunming mice (for each group, n = 6) were used and each mouse was challenged with 5-10 cercariae in infected group while another group served as non-infected control ( Figure S1). iScience Article LAMP-based CRISPR/Cas12a The LAMP reaction was performed with the listed primers (Table S4)  For CRISPR/Cas12a, crRNA was obtained using the MEGAscript T7 Transcription Kit (Thermofisher Scientific) using the below primers and annealed in vitro. The in vitro transcribed products were purified and the purified RNA was used as crRNA.

ELISA
ELISA was performed in a 96 well plate (Costar, USA) coated with 100 mL of egg antigens (10 mg/mL) with slight modification. 40 The 96-well plate (Costar, USA) was coated with 100 mL of SEA (10 mg/mL) in carbonate-bicarbonate buffer (pH 9.0) per well at 4 C overnight. Then, the plate was washed with PBS containing 0.05% Tween 20 (PBST) for three times and blocked with 100 mL 1% BSA in PBST at 37 C for 1 h. The plate was further washed with PBST and incubated with 100 mL serum diluted at 1:100 at 37 C for 1 h. The plate was washed with PBST and incubated with 100 mL horseradish peroxidase-conjugated goat anti-mouse IgG antibody diluted at 1:5000 at 37 C for 30 min. Following three washes, 100 mL 3,3 0 ,5,5 0 -tetramethyl benzidine dihydrochloride (TMB) was added to each well and incubated for 10 min. Then, 2M sulfuric acid was added to stop the reactions and the absorbance was determined using a microplate reader (BioTek, USA) at 450 nm.

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